Anti-vla-4 antibodies

ABSTRACT

This invention relates to alpha-4 binding antibodies, and fragments thereof.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/641,199, filed Oct. 15, 2012, which is a National Stage Applicationunder 35 U.S.C. § 371 from PCT/US2011/032641, filed Apr. 15, 2011, andclaims the benefit of U.S. Provisional Application No. 61/324,944, filedApr. 16, 2010, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to alpha-4 binding antibodies, and fragmentsthereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 9, 2012, isnamed B2047-7046US.txt and is 17,881 bytes in size.

BACKGROUND OF INVENTION

Humanized antibodies can be used as therapeutic agents in place ofmurine antibodies to avoid the undesirable immune response in humanstermed the HAMA (Human Anti-Mouse Antibody) response. Humanizedantibodies are generally constructed by replacing the complementarydetermining regions (CDRs) of a human antibody with the CDRs of anotherspecies, typically a mouse antibody.

VLA-4 (also called α4β1) is a member of the β1 integrin family of cellsurface receptors. VLA-4 contains an α4 chain and a β1 chain and isinvolved in cell-cell interactions. Its expression is mainly restrictedto lymphoid and myeloid cells. VLA-4 binds the endothelial cell ligandVCAM-1 (Vascular Cell Adhesion Molecule-1), and can mediate T and Blymphocyte attachment to the heparin II binding fragment of human plasmafibronectin.

SUMMARY OF INVENTION

The inventors have discovered that germline variable region frameworkscan be used to optimize CDR-grafted alpha-4 binding antibodies, such asanti-VLA-4 antibodies. Accordingly, the invention features anti-VLA-4variable heavy (VH) and variable light (VL) chains and antibodymolecules including such frameworks.

In one aspect, the invention features an anti-α4 antibody VH chainhaving CDRs from a donor anti-α4 antibody, e.g., an anti-α4 antibodydescribed herein, and a VH framework having regions 1, 2, 3, and 4 fromthe sequence of, or having no more than 5, 10 or 15 differences from agermline variable region sequence for the VH chain. In one embodiment,variable framework region 4 (FR4) is a human consensus sequence. In oneembodiment, the complete VH chain framework regions FR1, FR2, FR3 andFR4, are present. In another embodiment, the chain is an antigen-bindingfragment of a VH region.

In one embodiment, the germline sequence is human IGHV1-f (SEQ ID NO:2),depicted in FIG. 1. In certain embodiments, the VH framework sequencecan differ by at least one, but by no more than 2, 3, 4, 5, 10 or 15amino acid residues from a germline sequence, e.g., SEQ ID NO:2. In oneembodiment, the VH framework further includes other than thecorresponding human residues. For example, the VH chain includesnon-human residues, at one or more of framework positions 24, 67, 76,80, and 94 (Kabat numbering) of SEQ ID NO:2.

In one embodiment, at least one or more of the complementary determiningregions (CDRs) of the variable domains are derived from a donornon-human α4-binding antibody. In one embodiment, the antigen bindingregions of the CDR-grafted heavy chain variable domain include the CDRscorresponding to positions 26-34 (CDR1), 50-65 (CDR2) and 95-102 (CDR3)(Kabat numbering; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5^(th) ed., vol. 4, 1991, U.S. Department of Health and HumanServices, NIH, USA).

Thus, in one embodiment, the variable heavy chain (VH) framework has anacceptor sequence derived from human antibody germline sequence IGHV1-f.

In another embodiment, at least one amino acid, and less than 2, 3, 4,5, or 10 amino acid residues, in the FR1 region of the VH is other thanthe corresponding human germline residue. One or more of such residuescan, for example, be identical to the nonhuman antibody framework regionfrom which the CDR sequences are derived. In one embodiment, the aminoacid residue at Kabat position 24 is mutated to be identical to thenonhuman antibody framework region.

In another embodiment, at least one amino acid, and less than 2, 3, 4,5, or 10 amino acid residues, in the FR2 region of the VH is other thanthe corresponding human germline residue. One or more of such residuescan, for example, be identical to the nonhuman antibody framework regionfrom which the CDR sequences are derived.

In yet another embodiment, at least one amino acid, and less than 2, 3,4, 5, or 10 amino acid residues, in FR3 of the VH chain is other thanthe corresponding human germline residue. One or more of such residuescan, for example, be identical to the nonhuman antibody framework regionfrom which the CDR sequences are derived. In one embodiment, the aminoacid residue at Kabat position 94 is identical to the nonhuman antibodyframework region. In yet another embodiment, the amino acid residues atKabat positions 67, 76, 80, and 94 are identical to the nonhumanantibody framework region.

In certain embodiments, the VH chain of the antibody has the sequence ofSEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In one aspect, the invention features, an anti-VLA-4 VL chain havingCDRs from a donor anti-VLA-4 antibody, e.g., an anti-VLA-4 antibodydescribed herein, and a VL framework having regions 1, 2, 3, and 4 fromthe sequence of, or having no more than 5, 10 or 15 differences (eitherper/region or in total) from, a germline variable region sequence forthe VL chain. In one embodiment, variable framework region 4 (FR4) is ahuman consensus sequence. In one embodiment, the complete VL chainframework regions FR1, FR2, FR3 and FR4, are present. In anotherembodiment, the chain is an antigen-binding fragment of a VL region.

In another embodiment, the germline sequence is IGKV4-1 (SEQ ID NO:7),depicted in FIG. 2. In yet other embodiments, the VL framework sequencecan differ by at least one, but no more than 2, 3, 4, 5, 10 or 15 aminoacid residues from a germline framework sequence, e.g., SEQ ID NO:7. Inanother embodiment, the VL further includes other than the correspondinghuman amino acid residues. For example, the VL chain further includesnon-human residues at one or more of framework positions 1, 73, and 87(Kabat numbering) of SEQ ID NO:7.

In one embodiment, the sequence is AAH7035.1 (SEQ ID NO:12) or itsgermline engineered version (SEQ ID NO:13), depicted in FIG. 2. In someembodiments, the VL framework sequence can differ by at least one, butnot more than 5, 10, 15, 20, or 25 amino acid residues from a germlineengineered framework sequence, e.g., SEQ ID NO:13. In one embodiment,the VL chain includes other than the corresponding human residues. Forexample, the VL chain includes non-human residues at one or more offramework positions 1 and 87 (Kabat numbering) of SEQ ID NO:12. Inanother embodiment, the VL includes amino acid substitutions in theframework regions to resemble a different human germline frameworksequence, such as from germline sequence IGKV4-1. In certainembodiments, the VL framework sequence is altered to be identical to theIGKV4-1 germline sequence at positions 1-3, 5-23, 35-37, 39-42, 45-49,57, 59-61, 63-64, 70-72, 74-84, 86-88, 99-106 (Kabat numbering) of SEQID NO:12.

In one embodiment, at least one or more of the complementary determiningregions (CDRs) of the variable domains are derived from a donornon-human α4-binding antibody. In another embodiment, the antigenbinding regions of the CDR-grafted heavy chain variable domain includethe CDRs corresponding to positions 24-31 (CDR1), 50-56 (CDR2) and 89-97(CDR3) (Kabat numbering). Thus, in one embodiment, the VL framework hasan acceptor sequence constructed from IGKV4-1 germline sequence, fromantibody AAH70335.1 or from germline engineered antibody AAH70335.1.

In yet another embodiment, at least one amino acid, and less than 2, 3,4, 5, 10, or 15 residues, in FR1 of the VL chain is other than thecorresponding human residue. One or more of such residues can, forexample, be identical to the nonhuman antibody framework region fromwhich the CDR sequences are derived. In one embodiment, the amino acidresidue at the N-terminal position of FR1 is mutated to be identical tothe nonhuman antibody framework region.

In another embodiment, at least one amino acid, and less than 2, 3, 4,5, 10, or 15 residues, in FR2 of the VL chain is other than thecorresponding human residue. One or more of such residues can, forexample, be identical to the nonhuman antibody framework region fromwhich the CDR sequences are derived.

In yet another embodiment, at least one amino acid, and less than 2, 3,4, 5, 10, or 15 residues, in FR3 of the VL is other than thecorresponding human residue. One or more of such residues can, forexample, be identical to the nonhuman antibody framework region fromwhich the CDR sequences are derived. In another embodiment, the aminoacid residue at Kabat position 87 is mutated to be identical to thenonhuman antibody framework region. In yet another embodiment, the aminoacid residues at Kabat positions 67 and 87 are mutated to be identicalto the nonhuman antibody framework sequence. In yet another embodiment,the amino acid residues at Kabat positions 67, 73, and 87 of SEQ ID NO:7are mutated to be identical to the nonhuman antibody framework sequence.

In other embodiments, the VL chain of the antibody has the sequence ofSEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

In one embodiment, the CDRs of the VH and VL acceptor frameworksequences are selected to resemble the CDR sequences of a nonhuman(e.g., murine) antibody sequence, where the nonhuman antibody bindsintegrin alpha-4 or a fragment thereof. In another embodiment, thesequences of the CDRs are selected to resemble the sequences of the CDRsof a non-human antibody that binds the B1 epitope of the VLA-4 α4 chain.In one embodiment, the CDRs are selected to resemble a murine monoclonalantibody, e.g., HP1/2, HP2/1, HP2/4, L25, P4C2, or 21.6 (Pulido et al.,J. Biol. Chem. 266:10241-10245, 1991; U.S. Pat. No. 6,033,665).Modification can mean, e.g., excision and insertion or alteration, e.g.,by directed mutagenesis.

In another aspect, the invention features an antibody, or antigenbinding fragment thereof, including:

-   -   an anti-VLA-4 VL chain described herein, e.g., an anti-VLA-4 VL        chain having CDR's from a donor anti-VLA-4 antibody, e.g., an        anti-VLA-4 antibody described herein, and a VL framework having        LC framework regions 1, 2 and 3 from the sequence of, or having        no more than 5, 10, or 15 differences from, a germline variable        region sequence for the VL chain. In one embodiment, variable        region 4 is a human consensus sequence; and    -   an anti-VLA-4 VH chain described herein, e.g., an anti-VLA-4 VL        chain having CDRs from a donor anti-VLA-4 antibody, e.g., an        anti-VLA-4 antibody described herein, and a VL framework having        LC framework regions 1, 2 and 3 from the sequence of, or having        no more than 5, 10 or 15 differences from, a germline variable        region sequence for the VL chain. In one embodiment, variable        region 4 is a human consensus sequence.

In one embodiment, the antibody binds one or both of α4β1 and α4β7.

In another aspect, a VL or VH chain, or antibody, or fragment thereof,described herein is detectably labeled.

In yet another aspect, the invention features a vector containing DNAencoding an antibody heavy chain, or an α4 binding fragment thereof,described herein. In some embodiments, the DNA of the vector encodes aVH having the sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In yet another aspect, the invention features a vector containing DNAencoding an antibody light chain, or an α4 binding fragment thereof,described herein. In some embodiments, the DNA of the vector encodes aVL chain having the sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10or SEQ ID NO:11.

In yet another aspect, the invention features a vector containing DNAencoding an antibody heavy chain, or an α4 binding fragment thereof,described herein and an antibody light chain, or an α4 binding fragmentthereof, described herein.

In another aspect, the invention features a host cell containing avector described herein, e.g., one capable of expressing a heavy and/orlight chain antibody or antibody fragment described herein.

In one aspect, the invention features a method of making a recombinantanti-α4 antibody, or an α4-binding fragment thereof, by providing a hostcell transfected with (a) a DNA sequence encoding an antibody heavychain described herein, or an α4-binding fragment thereof, and (b) a DNAsequence encoding an antibody light chain, or an α4-binding fragmentthereof, and culturing the transfected cell to produce the recombinantanti-α4 antibody molecule or α4 binding fragment thereof. The DNAencoding the antibody heavy and light chains can be produced on the samevector or on different vectors.

In one aspect, the invention features a method of making a recombinantanti-α4 antibody, or an α4-binding fragment thereof, by providing a hostcell transfected with (a) a DNA sequence encoding an antibody heavychain, or an α4-binding fragment thereof, e.g., where the DNA sequencehas the sequence of SEQ ID NOs:3, 4, or 5, and (b) a DNA sequenceencoding an antibody light chain, or an α4-binding fragment thereof,e.g., wherein the DNA sequence has the sequence of SEQ ID NOs: 8, 9, 10,or 11, and culturing the transfected cell line to produce therecombinant anti-α4 antibody molecule or α4 binding fragment thereof.The DNA encoding the antibody heavy and light chains can be produced onthe same vector or on different vectors.

In another aspect, the invention features a method of treating a diseaseor disorder mediated by an α4 integrin, e.g., an α4β1 (VLA-4) or α4β7integrin, by administering an α4 antibody or antibody fragment describedherein, or a pharmaceutical composition containing the antibody orfragment, to a subject in need of such treatment. The subject can haveor be at risk for developing, for example, inflammatory, immune, orautoimmune disorders (e.g., inflammation of the central nervous system,such as multiple sclerosis, meningitis, neuromyelitis optica,neurosarcoidosis, CNS vasculitis, encephalitis, and transversemyelitis), tissue or organ graft rejection or graft-versus-host disease,acute CNS injury, such as stroke, traumatic brain injury (TBI), orspinal cord injury (SCI); chronic renal disease; allergy, e.g., allergicasthma; type 1 diabetes mellitus; inflammatory bowel disorders, such asCrohn's disease, ulcerative colitis; myasthenia gravis; fibromyalgia;arthritic disorders, such as rheumatoid arthritis, psoriatic arthritis;inflammatory/immune skin disorders, such as psoriasis, vitiligo,dermatitis, lichen planus; systemic lupus erythematosus; Sjogren'sSyndrome; hematological cancers, such as multiple myeloma, leukemia,lymphoma; solid cancers, such as sarcomas or carcinomas, e.g., of thelung, breast, prostate, brain; and fibrotic disorders, such as pulmonaryfibrosis, myelofibrosis, liver cirrhosis, mesangial proliferativeglomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy,and renal interstitial fibrosis.

In another aspect, the invention features a method of treating a patientby administering to the patient an α4-binding antibody or antibodyfragment. In one embodiment, the patient has a cancer, such as a solidtumor or a hematological malignancy. For example, a patient treated withan α4-binding antibody or antibody fragment can have acute myelogenousleukemia (AML) or multiple myeloma (MM).

In another embodiment, the patient has an inflammatory disorder, such asmultiple sclerosis, asthma (e.g., moderate to severe asthma), rheumatoidarthritis, diabetes, or Crohn's disease. In another embodiment, thecomposition is administered as a regimen. In yet another embodiment, themethod further includes selecting a patient suitable for treatment withthe composition. A patient suitable for treatment, for example, hasdemonstrated a sign or symptom indicative of disease onset, such as asign or symptom indicative of MS.

In yet another embodiment, the method further includes administering tothe patient a second therapeutic agent, such as, a chemotherapeuticagent, a thrombolytic agent, a neuroprotective agent, ananti-inflammatory agent, a steroid, a cytokine, or a growth factor.

In one embodiment, the patient is administered a humanized anti-VLA-4antibody, or fragment thereof, described herein, such as HuHP1/2, H1L1,H1L2 or H1L3.

In one embodiment, the composition containing an α4-binding antibody isadministered as a regimen, such at regular intervals. For example, thecomposition can be administered once daily, weekly or monthly; once perweek, twice per week, three times per week, four times per week or more;or once every two weeks, once every three weeks, once every four weeksor more.

In one embodiment, dosing can be adjusted according to a patient's rateof clearance of a prior administration of anti-α4 antibody. For example,in one embodiment, a patient will not be administered a second orfollow-on dose before the level of anti-α4 antibodies in the patient'ssystem has dropped below a pre-determined level. In one embodiment, asample from a patient (e.g., plasma, serum, blood or urine sample) isassayed for the presence of anti-α4 antibodies, and if the level ofanti-α4 antibodies is above a pre-determined level, the patient will notbe administered a second or follow-on dose. If the level of anti-α4antibodies in the patient's system is below a pre-determined level, thenthe patient is administered a second or follow-on dose.

In one embodiment, the composition is administered continuously, e.g.,over a period of more than 30 minutes but less than 1, 2, 4, or 12hours. The composition containing the antibody and the second agent canbe administered by any appropriate method, e.g., subcutaneously,intramuscularly, or intravenously.

In some embodiments, each of the antibody and the second agent isadministered at the same dose as each is prescribed for monotherapy. Inother embodiments, the antibody is administered at a dosage that isequal to or less than an amount required for efficacy if administeredalone. Likewise, the second agent can be administered at a dosage thatis equal to or less than an amount required for efficacy if administeredalone.

Another aspect featured in the disclosure is a method of evaluating apatient by determining if the patient meets a preselected criterion, andif the patient meets the preselected criterion approving, providing,prescribing, or administering a VLA-4 binding antibody formulationdescribed herein to the patient. In one embodiment, the preselectedcriterion is the failure of the patient to adequately respond to a prioralternate therapeutic treatment or regimen, e.g., for treatment of MS.In another embodiment, the preselected criterion is the absence of anysigns or symptoms of progressive multifocal leukoencephalopathy (PML),or the absence of any diagnosis of PML. In some cases, the selection isbased on the absence of a risk factor for PML, for example, the subjectdoes not test positive for JC virus DNA or does not test positive for JCvirus antibodies. In another embodiment, the criterion is as describedin PCT/US07/75577 (published as WO2008/021954), hereby incorporated byreference, which describes methods and systems for drug distribution andfor providing drugs to patients.

In another aspect, a method of distributing a composition describedherein is provided. The composition contains an alpha-4 bindingantibody. The method includes providing a recipient (e.g., an end user,patient, physician, retail or wholesale pharmacy, distributor, orpharmacy department at a hospital, nursing home clinic or HMO) with apackage containing sufficient unit dosages of the drug to treat apatient for at least 6, 12, 24, 36, or 48 months. In another aspect, theinvention features a method of evaluating the quality of a package orlot of packages (e.g., to determine if it has expired) of a compositiondescribed herein containing an alpha-4 binding antibody. The methodincludes evaluating whether the package has expired. The expiration dateis at least 6, 12, 24, 36, or 48 months, e.g., greater than 24 or 36months, from a preselected event, such as manufacturing, assaying, orpackaging. In some embodiments, a decision or step is taken as a resultof the analysis. For example, depending on the right analysis, theantibody in the package is used or discarded, classified, selected,released or withheld, shipped, moved to a new location, released intocommerce, sold, or offered for sale, withdrawn from commerce or nolonger offered for sale, depending on whether the product has expired.

In another aspect, the invention features a package containing at leasttwo unit doses of an aqueous composition containing an α4 bindingantibody. In one embodiment, all of the unit doses contain the sameamount of antibody, and in other embodiments there are unit dosages oftwo or more strengths, or two or more different formulations, e.g.,having different strengths or release properties.

In another aspect, the invention includes a method of instructing arecipient on the administration of a formulation containing α4 bindingantibody. The method includes instructing the recipient (e.g., an enduser, patient, physician, retail or wholesale pharmacy, distributor, orpharmacy department at a hospital, nursing home clinic or HMO) that theantibody should be administered to a patient according to a regimendescribed herein. The method can also include instructing the recipientthat the antibody should be administered prior to the expiration date.The expiration date is at least 6, 12, 24, 36, or 48 months, e.g.,greater than 24 or 36 months, from a preselected event, such asmanufacturing, assaying, or packaging. In one embodiment, the recipientalso receives a supply of the antibody, e.g., a supply of unit dosagesof the antibody.

In another aspect, the invention features a method of making an antibodywhich includes CDRs from a donor antibody, such as a non-human, e.g., amurine antibody, and one or both heavy and light chain variable regionframeworks derived from human germline variable region framework regionor regions. The method includes one or both of 1 and 2, where 1 and 2are as follows:

1. identifying or selecting a stable human acceptor heavy chain variableframework which has the same residues as the non-human donor heavy chainat one or more of the residues in one or more of a), b) and c):

a) VH Kabat #2, 4, 24, 26, 27, 29, 36, 38, 46, 47, 48, 49, 66, 67, 69,71, 78, 93, and 94, which, without being bound by theory, are believedto be important for maintaining CDR conformations;

b) VH Kabat #1, 2, 27, 28, 30, 43, 66, 68, 70, 72, 73, 74, and 75 which,without being bound by theory, are believed to be able to interact withantigen; and

c) VH Kabat #37, 39, 44, 45, 47, 91, 93 and 103, which, without beingbound by theory, are believed to be important for VH/VL interfaceintegrity; and

2. identifying or selecting a stable acceptor light chain variableframework which has the same residues as the donor light chain at one ormore of the residues in one or more of a), b) and c):

a) VL Kabat #2, 4, 38, 43, 44, 48, 58, 64, 71, and 73, which withoutbeing bound by theory, are believed to be important for maintaining CDRconformations;

b) VL Kabat #1, 2, 49, 57, 60, 63, 65, 66, 67, 68, 69, and 70 whichwithout being bound by theory, are believed to potentially be able tointeract with antigen; and

c) VL Kabat #36, 38, 43, 44, 46, 49, 87, and 98, which without beingbound by theory, are believed to be important for VH/VL interfaceintegrity;

3. providing a variable region having donor CDRs and the selectedgermline framework having matched residues identified in 1 or 2, such asby selecting a germline sequence and further backmutating additionalresidues identified in 1 or 2 of the germline to murine sequence so asto further maximize matching at the residues identified in 1 and 2; and

4. evaluating each matched position, such as by 3D structural analysisor modeling, and if a position meets a predetermined standard for riskof, for example, interfering with CDR conformations, antigeninteractions or VH/VL interface integrity, then reintroducing anequivalent murine residue, or a common human antibody residue,compatible with antibody structure. In one embodiment, at least 3, 4 or5 of the residues identified in (1.a) are matched. For example, in oneembodiment, residues 24, 29, or 94 are matched.

In one embodiment, at least 3, 4 or 5 of the residues identified in(1.b) are matched. For example, in one embodiment, residues 1, 73, or 75are matched.

In one embodiment, at least 3, 4 or 5 of the residues identified in(1.c) are matched. For example, in one embodiment, residues 37, 93, or103 are matched.

In one embodiment, at least 3, 4 or 5 of the residues identified in(2.a) are matched. For example, in one embodiment, residues 2, 71 and 73are matched.

In one embodiment, at least 3, 4 or 5 of the residues identified in(2.b) are matched. For example, in one embodiment, residues 1, 68, or 70are matched.

In one embodiment, at least 3, 4 or 5 of the residues identified in(2.c) are matched. For example, in one embodiment, residues 46, 87, or98 are matched.

In one embodiment, residue 6 in (1.a), residue 2 in (1.b), and residue 4in (1.c) are matched. In another embodiment, residue 4 in (2.a), residue2 in (2.b), and residue 4 in (2.c) are matched.

In one embodiment, the heavy chain germline sequence is of VH3, VH1 andVH5 germline class. In another embodiment, the light chain germlinesequence is a Vkappa or Vlambda sequence.

The term “treating” refers to administering a therapy in an amount,manner, and/or mode effective to improve a condition, symptom, orparameter associated with a disorder or to prevent progression of adisorder, to either a statistically significant degree or to a degreedetectable to one skilled in the art. An effective amount, manner, ormode can vary depending on the subject and may be tailored to thesubject.

An “α4 binding antibody” refers to an antibody that binds to the α4subunit of the VLA-4 (α4β1) integrin, and at least partially inhibits anactivity of VLA-4, particularly a binding activity of a VLA-4 integrinor a signaling activity, e.g., ability to transduce a VLA-4 mediatedsignal. For example, a VLA-4 binding antibody may inhibit binding ofVLA-4 to a cognate ligand of VLA-4, e.g., a cell surface protein such asVCAM-1 (Vascular Cell Adhesion Molecule-1), or to an extracellularmatrix component, such as fibronectin or osteopontin. An alpha-4 bindingantibody may bind to both α4β1 or α4β7. Typically, the antibody binds tothe B1 epitope of α4. An α4 binding antibody may bind to VLA-4 with aK_(d) of less than about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, or 10⁻¹¹ M.

As used herein, the term “antibody” refers to a protein that includes atleast one immunoglobulin variable region, e.g., an amino acid sequencethat provides an immunoglobulin variable domain or immunoglobulinvariable domain sequence. For example, an antibody can include a heavy(H) chain variable region (abbreviated herein as VH), and a light (L)chain variable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The light chains of the immunoglobulin may be oftypes kappa or lambda. In one embodiment, the antibody is glycosylated.An antibody can be functional for antibody dependent cytotoxicity and/orcomplement-mediated cytotoxicity, or may be non-functional for one orboth of these activities.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the FRs and CDRs has been precisely defined(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917). Kabat definitions are used herein. Each VH and VL istypically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxyl-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

An “immunoglobulin domain” refers to a domain from the variable orconstant domain of immunoglobulin molecules. Immunoglobulin domainstypically contain two β-sheets formed of about seven β-strands, and aconserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay(1988) Ann. Rev. Immunol. 6:381-405).

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence that can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may omit one, two or more N- or C-terminal aminoacids, internal amino acids, may include one or more insertions oradditional terminal amino acids, or may include other alterations. Inone embodiment, a polypeptide that includes an immunoglobulin variabledomain sequence can associate with another immunoglobulin variabledomain sequence to form a target binding structure (or “antigen bindingsite”), e.g., a structure that interacts with VLA-4.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains. The heavy and light immunoglobulin chains can be connected bydisulfide bonds. The heavy chain constant region typically includesthree constant domains, CH1, CH2 and CH3. The light chain constantregion typically includes a CL domain. The variable region of the heavyand light chains contains a binding domain that interacts with anantigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “immunoglobulin” comprises various broad classes ofpolypeptides that can be distinguished biochemically. Those skilled inthe art will appreciate that heavy chains are classified as gamma, mu,alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them(e.g., γ1-γ4). It is the nature of this chain that determines the“class” of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantinvention. All immunoglobulin classes are clearly within the scope ofthe present invention. Light chains are classified as either kappa orlambda (κ, λ). Each heavy chain class may be bound with either a kappaor lambda light chain.

The term “antigen-binding fragment” of a full length antibody refers toone or more fragments of a full-length antibody that retain the abilityto specifically bind to a target of interest, e.g., VLA-4. Examples ofbinding fragments encompassed within the term “antigen-binding fragment”of a full length antibody include (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂fragment, a bivalent fragment including two Fab fragments linked by adisulfide bridge at the hinge region; (iii) an Fd fragment consisting ofthe VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR) that retainsfunctionality. Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules known as single chain Fv (scFv). See e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883.

In some embodiments, the above-described antibodies are pegylated.

In some embodiments, the above-described antibodies or fragments thereofare multispecific. In further embodiments, the above-describedantibodies or fragments thereof are monovalent or bispecific.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 displays the three sequence variants of HP1/2 heavy chain (SEQ IDNO: 1)_to a human heavy germline IGHV1-f (SEQ ID NO: 2). The lower caseletters above the sequence represent insertions according to the Kabatnumbering scheme.

FIG. 2 displays the four sequence variants of HP1/2 light chain (SEQ IDNO: 6) to a germline IGKV4-1 antibody sequence (SEQ ID NO: 7) (DesignL0—SEQ ID NO: 8, L1—SEQ ID NO: 9, and L2—SEQ ID NO: 10) or human kappagermline engineered AAH7033.1 antibody sequence (SEQ ID NO: 12) (DesignL3—SEQ ID NO: 11). The lower case letters above the sequence representinsertions according to the Kabat numbering scheme.

FIG. 3 is a graph depicting the results of ELISA assays.

FIG. 4 is a graph depicting the results of ELISA assays.

FIG. 5 is the amino acid sequence of an IgG4 Fc (hinge+CH2+CH3domain)(SEQ ID NO:14). The hinge region is depicted in bold, and the CH3domain is underlined. The boxed “S” is Ser228. The circled “N” isAsn297.

FIG. 6 is a graph depicting flow cytometry data from binding of HuHP1/2to various tumor cell lines. “HP1/2” refers to humanized HP1/2.

FIGS. 7A-7C is a panel of graphs depicting inhibition of binding of AMLcell lines to fibronectin or VCAM1-Ig coated wells by HuHP1/2. FIG. 7Adepicts inhibition of binding of HL60 and KG1 cells to FN-coated wells.FIG. 7B depicts inhibition of binding of KG1 cells to VCAM1-Ig-coatedwells. FIG. 7C depicts inhibition of binding of HL60 cells to FN- andVCAM1-Ig-coated wells when incubated with 20 μg/mL HuHP1/2 (Solid bars).Clear bars indicate percent cell adhesion in the presence of an isotypecontrol. “HP1/2” refers to humanized HP1/2.

FIGS. 8A-8C make up a panel of graphs depicting inhibition of binding ofMM cell lines to fibronectin or VCAM1-Ig coated wells by HuHP1/2. FIG.8A depicts inhibition of binding of U266 and H929 cells to FN-coatedwells. FIG. 8B depicts inhibition of binding of U266 and H929 cells toVCAM1-Ig-coated wells. FIG. 8C depicts inhibition of binding of U266cells to FN- and VCAM1-Ig-coated wells when incubated with 20 μg/mLHuHP1/2 (Solid bars). Clear bars indicate percent cell adhesion in thepresence of an isotype control. “HP1/2” refers to humanized HP1/2.

FIGS. 9A-9C makes up a panel of graphs depicting inhibition of bindingof CLL cell lines to fibronectin or VCAM1-Ig coated wells by HuHP1/2.FIG. 9A depicts inhibition of binding of Mec1 and JM1 cells to FN-coatedwells. FIG. 9B depicts inhibition of binding of Mec1 and JM1 cells toVCAM1-Ig-coated wells. FIG. 9C depicts inhibition of binding of Mec1cells to FN- and VCAM1-Ig-coated wells when incubated with 20 μg/mLHuHP1/2 (Solid bars). Clear bars indicate percent cell adhesion in thepresence of an isotype control. “HP1/2” refers to humanized HP1/2.

DETAILED DESCRIPTION

Antibodies against VLA-4 have been demonstrated to be useful in treatingdisease. For example, natalizumab (TYSABRI®), an anti-VLA-4 antibody isused for treating relapsing multiple sclerosis and Crohn's disease.However, for treatment of certain conditions, for example acuteconditions such as spinal cord injury (SCI) or traumatic brain injury(TBI), or treatments that are administered in a finite number such astreatment of cancer, it may be advantageous to treat with an anti-VLA-4antibody that binds with an affinity different than natalizumab, e.g., ahigher affinity. In addition, treatment with anti-VLA-4 antibodies isassociated with a rare but sometimes fatal disorder, progressivemultifocal leukoencephalopathy (PML), for which a part of the treatmentrequires removal of antibody from the treated subject, for example usingplasma exchange or immunoabsorption. Because of the need to removeantibody, it is also desirable to balance the advantages of an antibodythat has increased affinity for VLA-4 with the disadvantage of anantibody that binds so tightly as to make removal difficult or to createa risk associated with a slow turnover rate. Such antibodies may also beuseful for treating conditions such as multiple sclerosis in that lessfrequent treatment may be required or administration by means other thaninfusion may be more efficient. Enabling treatment with lower doses mayalso lower the risk of adverse events such as PML. Accordingly, thepresent invention provides antibodies having such desirable properties.

The invention is based at least in part on the unexpectedcharacteristics of newly designed humanized α4-binding antibodies thathave a binding affinity for α4 that is 10-fold higher than that of theanti-α4 antibody natalizumab.

Alpha-4 binding antibodies, and fragments thereof, are provided wherethe variable light chain (VL) and variable heavy chain (VH) frameworkshave acceptor sequences constructed from germline or germline engineeredantibody sequences, such as IGKV4-1 or geAAH70335.1 or IGHV1-fantibodies. The CDR sequences are derived from nonhuman anti-α4 bindingantibodies such as the anti-VLA-4 antibody HP1/2. Antibodies describedherein can have an increase of at least 1.5, 2.0, 2.5, 3.0 fold inaffinity, e.g., relative to its murine parent. In one embodiment, theincrease in affinity is at least 1.5, 2.0, 2.5, 3.0 fold but isrespectively, less than 25, 20, or 15 fold.

Pharmaceutical Compositions

An α4 binding agent, such as a VLA-4 binding antibody, can be formulatedas a pharmaceutical composition. Typically, a pharmaceutical compositionincludes a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, and the like, aswell as from nontoxic organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, aromatic acids, aliphatic and aromatic sulfonic acids and thelike. Base addition salts include those derived from alkaline earthmetals, such as sodium, potassium, magnesium, calcium and the like, aswell as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

The antibody compositions described herein can be formulated accordingto methods known in the art. Pharmaceutical formulation is awell-established art, and is further described in Gennaro (ed.),Remington: The Science and Practice of Pharmacy, 20^(th) ed.,Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th) Ed.,Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); andKibbe (ed.), Handbook of Pharmaceutical Excipients AmericanPharmaceutical Association, 3^(rd) ed. (2000) (ISBN: 091733096X).

In one embodiment, the α4 antibody can be formulated with excipientmaterials, such as sodium chloride, sodium dibasic phosphateheptahydrate, sodium monobasic phosphate, and polysorbate 80. In anotherembodiment, the α4 antibody can be formulated in a citrate buffer, e.g.,at pH 5, 5.5, 6, 6.5, 7, or 7.5. In yet another embodiment, the α4antibody can be formulated in a solution including 2, 4, 5, 6, 8, 10,12, 14, or 15% sucrose. It can be provided, for example, in a bufferedsolution at a concentration of about 20 mg/ml and can be stored at 2-8°C.

Pharmaceutical compositions may also be in a variety of other forms.These include, for example, liquid, semi-solid and solid dosage forms,such as liquid solutions (e.g., injectable and infusible solutions),dispersions or suspensions, tablets, pills, powders, liposomes andsuppositories. The form can depend on the intended mode ofadministration and therapeutic application. Typically, compositions forthe agents described herein are in the form of injectable or infusiblesolutions.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. A pharmaceutical compositioncan also be tested to insure it meets regulatory and industry standardsfor administration.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating an agent described herein in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating an agent described herein intoa sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,typical methods of preparation are vacuum drying and freeze-drying thatyields a powder of an agent described herein plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Administration

An α4 binding antibody can be administered to a subject, e.g., a humansubject, by a variety of methods. For many applications, the route ofadministration is one of: intravenous injection or infusion,subcutaneous injection, or intramuscular injection. An α4 bindingantibody can be administered as a fixed dose, or in a mg/kg dose. Theantibody can be administered intravenously (IV) or subcutaneously (SC).For example, the antibody can be administered at a fixed unit dose ofbetween about 50-600 mg IV, e.g., every 4 weeks, or between about 50-100mg SC (e.g., 75 mg), e.g., at least once a week (e.g., twice a week). Inone embodiment, the antibody is administered IV at a fixed unit dose of50 mg, 60 mg, 80 mg, 100 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 180mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg or more. Administration ofthe IV dose can be once or twice or three times or more per week, oronce every two, three, four, or five weeks, or less frequently.

In one embodiment, the antibody is administered SC at a fixed unit doseof 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 100 mg, or 120 mg or more.Administration of the SC dose can be once or twice or three times ormore per week, or once every two, three, four, or five weeks, or lessfrequently.

An anti-α4 antibody can also be administered in a bolus at a dose ofbetween about 1 and 10 mg/kg, e.g., about 6.0 mg/kg, 4.0 mg/kg, 3.0mg/kg, 2.0 mg/kg, 1.0 mg/kg. Modified dose ranges include a dose that isless than about 600 mg/subject, about 400 mg/subject, about 300mg/subject, about 250 mg/subject, about 200 mg/subject, or about 150mg/subject, typically for administration every fourth week or once amonth. The α4 binding antibody can be administered, for example, everythree to five weeks, e.g., every fourth week, or monthly.

Dosing can be adjusted according to a patient's rate of clearance of aprior administration of anti-α4 antibody. For example, a patient may notbe administered a second or follow-on dose before the level of anti-α4antibodies in the patient's system has dropped below a pre-determinedlevel. In one embodiment, a sample from a patient (e.g., plasma, serum,blood, urine, or cerebrospinal fluid (CSF)) is assayed for the presenceof anti-α4 antibodies, and if the level of anti-α4 antibodies is above apre-determined level, the patient will not be administered a second orfollow-on dose. If the level of anti-α4 antibodies in the patient'ssystem is below a pre-determined level, then the patient is administereda second or follow-on dose. A patient whose anti-α4 levels aredetermined to be too high (above the pre-determined level) can be testedagain after one or two or three days, or a week, and if the level ofanti-α4-antibody in the patient samples has dropped below thepre-determined level, the patient may be administered a second orfollow-on dose of antibody.

The dose can also be chosen to reduce or avoid production of antibodiesagainst the α4 binding antibody, to achieve greater than 40, 50, 70, 75,or 80% saturation of the α4 subunit, to achieve less than 80, 70, 60,50, or 40% saturation of the α4 subunit, or to prevent an increase inthe level of circulating white blood cells

In certain embodiments, the active agent may be prepared with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known. See, e.g., Controlled Drug Delivery (Drugs and thePharmaceutical Sciences), Second Edition, J. Robinson and V. H. L. Lee,eds., Marcel Dekker, Inc., New York, 1987.

Pharmaceutical compositions can be administered with a medical device.For example, pharmaceutical compositions can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;4,790,824; or 4,596,556. Examples of well-known implants and modules arediscussed in, e.g., U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multichamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Of course,many other such implants, delivery systems, and modules are also known.

This disclosure also features a device for administering a first andsecond agent. The device can include, for example, one or more housingsfor storing pharmaceutical preparations, and can be configured todeliver unit doses of the first and second agent. The first and secondagents can be stored in the same or separate compartments. For example,the device can combine the agents prior to administration. It is alsopossible to use different devices to administer the first and secondagent.

Dosage regimens are adjusted to provide the desired response, such as atherapeutic response or a combinatorial therapeutic effect. Generally,any combination of doses (either separate or co-formulated) of the VLA-4binding agent and the second agent can be used in order to provide asubject with both agents in bioavailable quantities.

Dosage unit form or “fixed dose” as used herein refers to physicallydiscrete units suited as unitary dosages for the subjects to be treated;each unit contains a predetermined quantity of active compoundcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier and optionally in association withthe other agent.

A pharmaceutical composition may include a “therapeutically effectiveamount” of an agent described herein. Such effective amounts can bedetermined based on the combinatorial effect of the administered firstand second agent. A therapeutically effective amount of an agent mayalso vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the compound to elicit adesired response in the individual, such as amelioration of at least onedisorder parameter, e.g., a multiple sclerosis parameter, oramelioration of at least one symptom of the disorder, e.g., a symptom ofmultiple sclerosis, such as muscle atrophy, ataxia, and tremors. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

Devices and Kits

Formulations containing an antibody described herein can be administeredwith a medical device. The device can be designed with features such asportability, room temperature storage, and ease of use so that it can beused in emergency situations, such as by an untrained subject or byemergency personnel in the field, removed to medical facilities andother medical equipment. The device can include, for example, one ormore housings for storing pharmaceutical preparations that include anα4-binding antibody, and can be configured to deliver one or more unitdoses of the agent.

For example, the pharmaceutical composition can be administered with atranscutaneous delivery device, such as a syringe, including ahypodermic or multichamber syringe. Other suitable delivery devicesinclude stents, catheters, microneedles, and implantable controlledrelease devices. The composition can be administered intravenously withstandard IV equipment, including, e.g., IV tubings, with or withoutin-line filters. In certain embodiments, the device will be a syringefor use in SC or IM administration.

Pharmaceutical compositions can be administered with medical devices.For example, pharmaceutical compositions can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;4,790,824; or 4,596,556. Examples of well-known implants and modules aredescribed in, e.g., U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multichamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thetherapeutic composition can also be in the form of a biodegradable ornonbiodegradable sustained release formulation for subcutaneous orintramuscular administration. Methods for such compositions are known inthe art. Continuous administration can also be achieved using animplantable or external pump. The administration can also be conductedintermittently, such as by single daily injection, or continuously at alow dose, such as in a sustained release formulation. The deliverydevice can be modified to be optimally suited for administration of anα4-binding antibody. For example, a syringe can be siliconized to anextent that is optimal for storage and delivery of the antibody. Ofcourse, many other such implants, delivery systems, and modules are alsoknown.

This disclosure also features a device for administering a first andsecond agent (e.g., an antibody and a second agent). The device caninclude, for example, one or more housings for storing pharmaceuticalpreparations, and can be configured to deliver unit doses of the firstand second agent. The first and second agents can be stored in the sameor separate compartments. In one embodiment, the device combines theagents prior to administration. In some embodiments, the first andsecond agents are administered by different devices.

An α4-binding antibody can be provided in a kit. In one embodiment, thekit includes (a) a container that contains a composition that includes ahigh concentration of VLA-4-binding antibody, optionally (b) a containerthat contains a composition that includes a second agent, and optionally(c) informational material. The informational material can bedescriptive, instructional, marketing or other material that relates tothe methods described herein and/or the use of the agents fortherapeutic benefit. In one embodiment, the kit also includes a secondagent. For example, the kit includes a first container that contains acomposition that includes the α4-binding antibody, and a secondcontainer that includes the second agent.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the antibody, concentration, date of expiration, batch orproduction site information, and so forth. In one embodiment, theinformational material relates to methods of administering theα4-binding antibody, e.g., in a suitable dose, dosage form, or mode ofadministration (e.g., a dose, dosage form, or mode of administrationdescribed herein), to treat a subject who has an acute disorder such asa spinal cord injury or traumatic brain injury, or an inflammatorydisease (e.g., MS), or who is at risk for experiencing an episodeassociated with an inflammatory disease. The information can be providedin a variety of formats, including printed text, computer readablematerial, video recording, or audio recording, or information thatprovides a link or address to substantive material.

In addition to the agent, the composition in the kit can include otheringredients, such as a solvent or buffer, a stabilizer, or apreservative. The agent can be provided in any form, e.g., liquid, driedor lyophilized form, and substantially pure and/or sterile. When theagents are provided in a liquid solution, the liquid solution typicallyis an aqueous solution. When the agents are provided as a dried form,reconstitution generally is by the addition of a suitable solvent. Thesolvent, e.g., sterile water or buffer, can optionally be provided inthe kit.

The kit can include one or more containers for the composition orcompositions containing the agents. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theagents. The containers can include a combination unit dosage, e.g., aunit that includes both the α4 binding antibody and the second agent,such as in a desired ratio. For example, the kit can include a pluralityof syringes, ampoules, foil packets, blister packs, or medical deviceseach containing, for example, a single combination unit dose. Thecontainers of the kits can be air tight, waterproof (e.g., impermeableto changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administering thecomposition, e.g., a syringe or other suitable delivery device. Thedevice can be provided pre-loaded with one or both of the agents or canbe empty but suitable for loading.

Oncology

The α4-binding antibodies and methods described herein can be used totreat cancer, including solid cancers and hematological malignancies.Exemplary solid cancers include sarcomas and carcinomas, such as of thelung, breast, pancreas, colon, prostate, bladder and brain.Hemotological malignancies include cancers such as multiple myeloma,leukemia, and lymphoma.

Methods are provided for treating a patient having a hematologicalmalignancy with a composition containing an α4-binding antibody, such asanti-VLA-4 antibody described herein. Hematological malignancies arecancers of the body's blood-forming and immune systems. Cancers of thistype affect the blood, bone marrow, and/or lymph nodes. Hematologicalmalignancies include leukemias, such as acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia(CML), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia,acute erythroleukemia, and hairy cell leukemia (HCL); lymphomas, such asHodgkin's disease and Non-Hodgkin's lymphoma; and multiple myeloma;Waldenstrom's macroblobulinemia; myelodysplastic syndrome (MDS) (whichcan culminate in AML); a myeloproliferative disease, such aspolycythemia vera (also called PV, PCV or polycythemia rubra vera(PRV)), Essential thrombocytosis (ET), myelofibrosis, heavy chaindisease; and amyloid due to light-chain disease.

Patients having a hematological malignancy may be identified by analysisof blood count and blood film by, for example, light microscopy, whichis useful for identifying malignant cells. A biopsy, such as from bonemarrow, can also be used to identify malignant cells, and a biopsy froma lymph node can be useful for identifying a lymphadenopathy.

An α4-binding antibody (e.g., a humanized anti-VLA-4 antibody, such asHuHP1/2, H1L0, H1L1, H1L2 or H1L3) is useful for the treatment of aleukemia, such as AML. Leukemias are cancers that originate in the bonemarrow, where the malignant cells are white blood cells (leukocytes).AML (also called acute myelocytic leukemia, acute myeloblastic leukemia,acute granulocytic leukemia, and acute nonlymphocytic leukemia) is amalignancy that arises in either granulocytes or monocytes. AML ischaracterized by the uncontrolled, exaggerated growth and accumulationof cells called leukemic blasts, which fail to function as normal bloodcells, and the blockade of the production of normal marrow cells,leading to a deficiency of red cells (anemia), and platelets(thrombocytopenia) and normal white cells (especially neutrophils, i.e.,neutropenia) in the blood.

All subtypes of AML are suitable for treatment with a VLA-4 bindingantibody. The subtypes of AML are classified based on the stage ofdevelopment myeloblasts have reached at the time of diagnosis. Thecategories and subsets allow the physician to decide what treatmentworks best for the cell type and how quickly the disease may develop.The subsets are: M0, myeloblastic, on special analysis; M1,Myeloblastic, without maturation; M2, Myeloblastic, with maturation; M3,Promyelocytic; M4, Myelomonocytic; M5, Monocytic; M6, Erythroleukemia;and M7, Megakaryocytic. A VLA-4 antibody can be administered with asecondary agent that is particularly suited to the subtype of AML. Forexample, acute promyelocytic leukemia (APL) and acute monocytic leukemiaare subtypes of AML that need different treatment than other subtypes ofAML. A second agent for treatment of APL can include all-trans retinoicacid (ATRA) or an antimetabolite, such as cytarabine. A second agent fortreatment of acute monocytic leukemia can include a deoxyadenosineanalog, such as 2-chloro-2′-deoxyadenosine (2-CDA).

Risk factors of AML include the presence of certain genetic disorders,such as Down syndrome, Fanconi anemia, Shwachman-Diamond syndrome andothers. A patient having AML and a genetic disorder can be administereda VLA-4 binding antibody and a second agent to treat a symptom of thegenetic disorder. For example, a patient with AML and Fanconi anemia canbe administered a VLA-4 binding antibody and an antibiotic.

Other risk factors for AML include chemotherapy or radiotherapy fortreatment of a different cancer, tobacco smoke, and exposure to largeamounts of benzene.

Other cancers suitable for treatment with an α4-binding antibodyinclude, solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, ovarian cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular cancer, small cell lung carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, schwannoma, meningioma, melanoma,neuroblastoma, and retinoblastoma).

Other Disorders

The formulations and methods described herein can also be used to treatother inflammatory, immune, or autoimmune disorders, e.g., inflammationof the central nervous system (e.g., in addition to multiple sclerosis,meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis,encephalitis, and transverse myelitis); tissue or organ graft rejectionor graft-versus-host disease; acute CNS injury, e.g., stroke or spinalcord injury (SCI); chronic renal disease; allergy, e.g., allergicasthma, moderate to severe allergic rhinitis, ocular allergy; type 1diabetes mellitus; inflammatory bowel disorders, e.g., Crohn's disease,ulcerative colitis (e.g., for treatment or maintenance of remission);eosinophilic gastroenteritis; myasthenia gravis; fibromyalgia; disordersassociated with rheumatology/immunology, such as arthritic disorders,e.g., rheumatoid arthritis, psoriatic arthritis; dermatologicaldisorders, such as inflammatory/immune skin disorders, e.g., psoriasis,vitiligo, dermatitis (e.g., atopic dermatitis), lichen planus, moderateto severe chronic urticaria; systemic lupus erythematosus (SLE; e.g.,lupus nephritis); scleroderma (e.g., Progressive Systemic Sclerosis(PSS), such as PSS of the lung); acute or chronic primary eosinophilicpneumonia; Sjogren's Syndrome; acute coronary syndrome (ACS); acutemyocardial infarction; atherosclerosis; and fibrotic disorders, e.g.,pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), lung fibrosis(e.g., XRT induced), myelofibrosis, liver cirrhosis, mesangialproliferative glomerulonephritis, crescentic glomerulonephritis,diabetic nephropathy, and renal interstitial fibrosis.

The formulations and methods described herein can also be used to treatneurological disorders, such as cerebral ischemia, including preventionin patients with transient ischemic attacks and/or arterial stenosis.Other exemplary neurological disorders include chronic inflammatorydemyelinating polyneuropathy (CIDP); Guillian-Barre Syndrome (GBS);ocular diseases, such as macular degeneration (e.g., wet maculardegeneration), and anteriorischemic optic neuropathy; neuropathic pain(e.g., symptomatic neuropathic pain); Alzheimer's Disease; AmyotrophicLateral Sclerosis (ALS) (e.g., disease modifying ALS)′ and Parkinson'sDisease.

The formulations and methods described herein can also be used to treatpatients who have undergone transplantation, such as renal, heart, orbone marrow transplantation.

Multiple Sclerosis

Formulations containing an alpha-4 binding antibody described herein areuseful for the treatment of inflammatory diseases, such as multiplesclerosis (MS). Multiple sclerosis is a central nervous system diseasethat is characterized by inflammation and loss of myelin sheaths.

Patients having MS may be identified by criteria establishing adiagnosis of clinically definite MS as defined by the workshop on thediagnosis of MS (Poser et al., Ann. Neurol. 13:227, 1983). For example,an individual with clinically definite MS has had two attacks andclinical evidence of either two lesions or clinical evidence of onelesion and paraclinical evidence of another, separate lesion. DefiniteMS may also be diagnosed by evidence of two attacks and oligoclonalbands of IgG in cerebrospinal fluid or by combination of an attack,clinical evidence of two lesions and oligoclonal band of IgG incerebrospinal fluid. The McDonald criteria can also be used to diagnoseMS. (McDonald et al., 2001, “Recommended diagnostic criteria formultiple sclerosis: guidelines from the International Panel on theDiagnosis of Multiple Sclerosis,” Ann. Neurol. 50:121-127). The McDonaldcriteria include the use of MRI evidence of CNS impairment over time tobe used in diagnosis of MS, in the absence of multiple clinical attacks.Effective treatment of multiple sclerosis may be evaluated in severaldifferent ways. The following parameters can be used to gaugeeffectiveness of treatment. Two exemplary criteria include: EDSS(extended disability status scale), and appearance of exacerbations onMRI (magnetic resonance imaging). The EDSS is a method for gradingclinical impairment due to MS (Kurtzke, Neurology 33:1444, 1983). Eightfunctional systems are evaluated for the type and severity of neurologicimpairment. Briefly, prior to treatment, patients are evaluated forimpairment in the following systems: pyramidal, cerebella, brainstem,sensory, bowel and bladder, visual, cerebral, and other. Follow-ups areconducted at defined intervals. The scale ranges from 0 (normal) to 10(death due to MS). A decrease of one full step indicates an effectivetreatment (Kurtzke, Ann. Neurol. 36:573-79, 1994). Patients may also bediagnosed using other criteria used by those in the art.

Exacerbations are defined as the appearance of a new symptom that isattributable to MS and accompanied by an appropriate new neurologicabnormality (IFNB MS Study Group, supra). In addition, the exacerbationmust last at least 24 hours and be preceded by stability or improvementfor at least 30 days. Briefly, patients are given a standardneurological examination by clinicians. Exacerbations are either mild,moderate, or severe according to changes in a Neurological Rating Scale(Sipe et al., Neurology 34:1368, 1984). An annual exacerbation rate andproportion of exacerbation-free patients are determined.

Therapy can be deemed to be effective if there is a statisticallysignificant difference in the rate or proportion of exacerbation-free orrelapse-free patients between the treated group and the placebo groupfor either of these measurements. In addition, time to firstexacerbation and exacerbation duration and severity may also bemeasured. A measure of effectiveness as therapy in this regard is astatistically significant difference in the time to first exacerbationor duration and severity in the treated group compared to control group.An exacerbation-free or relapse-free period of greater than one year, 18months, or 20 months is particularly noteworthy. Efficacy may also beassessed using any method used in the art, for example to assesssymptoms of MS, including mobility improvement using a timed walk testused alone or in combination with other criteria,

Efficacy of administering a first agent and, optionally, a second agent,can also be evaluated based on one or more of the following criteria:frequency of MBP reactive T cells determined by limiting dilution,proliferation response of MBP reactive T cell lines and clones, cytokineprofiles of T cell lines and clones to MBP established from patients.Efficacy is indicated by decrease in frequency of reactive cells, areduction in thymidine incorporation with altered peptide compared tonative, and a reduction in TNF and IFN-α.

Clinical measurements include the relapse rate in one and two-yearintervals, and a change in EDSS, including time to progression frombaseline of 1.0 unit on the EDSS that persists for six months. On aKaplan-Meier curve, a delay in sustained progression of disability showsefficacy. Other criteria include a change in area and volume of T2images on MRI, and the number and volume of lesions determined bygadolinium enhanced images.

MRI can be used to measure active lesions using gadolinium-DTPA-enhancedimaging (McDonald et al. Ann. Neurol. 36:14, 1994) or the location andextent of lesions using T₂-weighted techniques. Briefly, baseline MRIsare obtained. The same imaging plane and patient position are used foreach subsequent study. Positioning and imaging sequences can be chosento maximize lesion detection and facilitate lesion tracing. The samepositioning and imaging sequences can be used on subsequent studies. Thepresence, location and extent of MS lesions can be determined byradiologists. Areas of lesions can be outlined and summed slice by slicefor total lesion area. Three analyses may be done: evidence of newlesions, rate of appearance of active lesions, percentage change inlesion area (Paty et al., Neurology 43:665, 1993). Improvement due totherapy can be established by a statistically significant improvement inan individual patient compared to baseline or in a treated group versusa placebo group.

Exemplary symptoms associated with multiple sclerosis, which can betreated with the methods described herein, include: optic neuritis,diplopia, nystagmus, ocular dysmetria, internuclear ophthalmoplegia,movement and sound phosphenes, afferent pupillary defect, paresis,monoparesis, paraparesis, hemiparesis, quadraparesis, plegia,paraplegia, hemiplegia, tetraplegia, quadraplegia, spasticity,dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus,myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctionalreflexes, paraesthesia, anaesthesia, neuralgia, neuropathic andneurogenic pain, l'hermitte's, proprioceptive dysfunction, trigeminalneuralgia, ataxia, intention tremor, dysmetria, vestibular ataxia,vertigo, speech ataxia, dystonia, dysdiadochokinesia, frequentmicturation, bladder spasticity, flaccid bladder, detrusor-sphincterdyssynergia, erectile dysfunction, anorgasmy, frigidity, constipation,fecal urgency, fecal incontinence, depression, cognitive dysfunction,dementia, mood swings, emotional lability, euphoria, bipolar syndrome,anxiety, aphasia, dysphasia, fatigue, uhthoffs symptom, gastroesophagealreflux, and sleeping disorders.

Each case of MS displays one of several patterns of presentation andsubsequent course. Most commonly, MS first manifests itself as a seriesof attacks followed by complete or partial remissions as symptomsmysteriously lessen, only to return later after a period of stability.This is called relapsing-remitting (RR) MS. Primary-progressive (PP) MSis characterized by a gradual clinical decline with no distinctremissions, although there may be temporary plateaus or minor relieffrom symptoms. Secondary-progressive (SP) MS begins with arelapsing-remitting course followed by a later primary-progressivecourse. Rarely, patients may have a progressive-relapsing (PR) course inwhich the disease takes a progressive path punctuated by acute attacks.PP, SP, and PR are sometimes lumped together and called chronicprogressive MS.

A few patients experience malignant MS, defined as a swift andrelentless decline resulting in significant disability or even deathshortly after disease onset. This decline may be arrested or deceleratedby administration of a combination therapy described herein.

Administration of an anti-α4 antibody featured herein can be effectiveto relieve one or more symptoms of MS, such as one or more of thesymptoms described above. For example, administration of an anti-α4antibody described herein can be used to treat primary or secondaryprogressive multiple sclerosis (PPMS or SPMS, respectively), andtreatment with an anti-α4 antibody can be effective to prevent relapse.

In addition to or prior to human studies, an animal model can be used toevaluate the efficacy of using the two agents. An exemplary animal modelfor multiple sclerosis is the experimental autoimmune encephalitis (EAE)mouse model, e.g., as described in (Tuohy et al. (J. Immunol. (1988)141: 1126-1130), Sobel et al. (J. Immunol. (1984) 132: 2393-2401), andTraugott (Cell Immunol. (1989) 119: 114-129). Mice can be administered afirst and second agent described herein prior to EAE induction. Then themice are evaluated for characteristic criteria to determine the efficacyof using the two agents in the model.

Antibody Generation

Recombinant antibodies that bind to alpha-4 can be generated by in vivoor in vitro methods such as phage display. The methods can be used tosupply anti-α4 CDRs for use in CDR grafted antibodies described herein.In addition, methods such as phage display can be used to select suchCDRs in the context of the germline frameworks disclosed herein, such asby using a library where the framework is a germline framework.

EP 239 400 (Winter et al.) describes altering antibodies by substitution(within a given variable region) of their complementarity determiningregions (CDRs) for one species with those from another. CDR-substitutedantibodies can be less likely to elicit an immune response in humanscompared to true chimeric antibodies because the CDR-substitutedantibodies contain considerably less non-human components. (Riechmann etal., 1988, Nature 332, 323-327; Verhoeyen et al., 1988, Science 239,1534-1536). Typically, CDRs of a murine antibody substituted into thecorresponding regions in a human antibody by using recombinant nucleicacid technology to produce sequences encoding the desired substitutedantibody. Human constant region gene segments of the desired isotype(usually gamma I for CH and kappa for CL) can be added and the heavy andlight chain genes can be co-expressed in mammalian cells to producesoluble antibody. Large nonimmunized phage display libraries may also beused to isolate high affinity antibodies that can be developed as humantherapeutics using standard phage technology (see, e.g., Hoogenboom etal. (1998) Immunotechnology 4:1-20; and Hoogenboom et al. (2000) ImmunolToday 2:371-8; U.S. 2003-0232333).

An anti-α4 antibody or antibody fragment described herein can recognizeepitopes of the α4 subunit that are involved in binding to a cognateligand, e.g., VCAM-1 or fibronectin. The antibodies described herein caninhibit binding of to one or more of the cognate ligands (e.g., VCAM-1and fibronectin).

In some embodiments, the antibodies featured herein, can interact withVLA-4 on cells, e.g., lymphocytes, but do not cause cell aggregation.

An exemplary α4 binding antibody has one or more CDRs, e.g., all threeheavy chain (HC) CDRs and/or all three light chain (LC) CDRs of aparticular antibody disclosed herein, or CDRs that are, in sum, at least80, 85, 90, 92, 94, 95, 96, 97, 98, 99% identical to such an antibody.In one embodiment, the H1 and H2 hypervariable loops have the samecanonical structure as those of an antibody described herein. In oneembodiment, the L1 and L2 hypervariable loops have the same canonicalstructure as those of an antibody described herein.

In one embodiment, the amino acid sequence of the HC and/or LC variabledomain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100%identical to the amino acid sequence of the HC and/or LC variable domainof an antibody described herein. The amino acid sequence of the HCand/or LC variable domain sequence can differ by at least one aminoacid, but no more than ten, eight, six, five, four, three, or two aminoacids from the corresponding sequence of an antibody described herein.For example, the differences may be primarily or entirely in theframework regions.

The amino acid sequences of the HC and LC variable domain sequences canbe encoded by a nucleic acid sequence that hybridizes under highstringency conditions to a nucleic acid sequence described herein or onethat encodes a variable domain or an amino acid sequence describedherein. In one embodiment, the amino acid sequences of one or moreframework regions (e.g., FR1, FR2, FR3, and/or FR4) of the HC and/or LCvariable domain are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100%identical to corresponding framework regions of the HC and LC variabledomains of an antibody described herein. In one embodiment, one or moreheavy or light chain framework regions (e.g., HC FR1, FR2, and FR3) areat least 70, 80, 85, 90, 95, 96, 97, 98, or 100% identical to thesequence of corresponding framework regions from a human germlineantibody.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences.

As used herein, the term “hybridizes under high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. High stringency hybridizationconditions include hybridization in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 65° C., or substantiallysimilar conditions.

Antibody Production

Antibodies can be produced in prokaryotic and eukaryotic cells. In oneembodiment, the antibodies (e.g., scFvs) are expressed in a yeast cellsuch as Pichia (see, e.g., Powers et al. (2001) J. Immunol. Methods251:123-35), Hanseula, or Saccharomyces.

In one embodiment, antibodies, particularly full length antibodies,e.g., IgGs, are produced in mammalian cells. Exemplary mammalian hostcells for recombinant expression include Chinese Hamster Ovary (CHOcells) (including dhfr− CHO cells, described in Urlaub and Chasin (1980)Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectablemarker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol.159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2cells, COS cells, K562, and a cell from a transgenic animal, e.g., atransgenic mammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the immunoglobulindomain, the recombinant expression vectors may carry additional nucleicacid sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017). Exemplary selectable markergenes include the dihydrofolate reductase (DHFR) gene (for use in dhfr⁻host cells with methotrexate selection/amplification) and the neo gene(for G418 selection).

In an exemplary system for recombinant expression of an antibody (e.g.,a full length antibody or an antigen-binding portion thereof), arecombinant expression vector encoding both the antibody heavy chain andthe antibody light chain is introduced into dhfr− CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, to transfect the host cells, to selectfor transformants, to culture the host cells, and to recover theantibody from the culture medium. For example, some antibodies can beisolated by affinity chromatography with a Protein A or Protein G. Forexample, purified α4-binding antibodies can be concentrated to about 100mg/mL to about 200 mg/mL using protein concentration techniques that areknown in the art.

Antibodies may also include modifications, e.g., modifications thatalter Fc function, e.g., to decrease or remove interaction with an Fcreceptor or with C1q, or both. For example, the human IgG4 constantregion can have a Ser to Pro mutation at residue 228 to fix the hingeregion. The amino acid sequence of an IgG4 Fc (hinge+CH2+CH3 domain) isprovided in FIG. 5.

In another example, the human IgG1 constant region can be mutated at oneor more residues, e.g., one or more of residues 234 and 237, e.g.,according to the numbering in U.S. Pat. No. 5,648,260. Other exemplarymodifications include those described in U.S. Pat. No. 5,648,260.

For some antibodies that include an Fc domain, the antibody productionsystem may be designed to synthesize antibodies in which the Fc regionis glycosylated. In another example, the Fc domain of IgG molecules isglycosylated at asparagine 297 in the CH2 domain (see FIG. 5). Thisasparagine is the site for modification with biantennary-typeoligosaccharides. This glycosylation participates in effector functionsmediated by Fcγ receptors and complement C1q (Burton and Woof (1992)Adv. Immunol. 51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76).The Fc domain can be produced in a mammalian expression system thatappropriately glycosylates the residue corresponding to asparagine 297.The Fc domain can also include other eukaryotic post-translationalmodifications.

Other suitable Fc domain modifications include those described inWO2004/029207. For example, the Fc domain can be an XMAB® Fc (Xencor,Monrovia, Calif.). The Fc domain, or a fragment thereof, can have asubstitution in an Fcγ Receptor (FcγR) binding region, such as thedomains and fragments described in WO05/063815. In some embodiments, theFc domain, or a fragment thereof, has a substitution in a neonatal FcReceptor (FcRn) binding region, such as the domains and fragmentsdescribed in WO05047327. In other embodiments, the Fc domain is a singlechain, or fragment thereof, or modified version thereof, such as thosedescribed in WO2008143954. Other suitable Fc modifications are known anddescribed in the art.

Antibodies can also be produced by a transgenic animal. For example,U.S. Pat. No. 5,849,992 describes a method for expressing an antibody inthe mammary gland of a transgenic mammal. A transgene is constructedthat includes a milk-specific promoter and nucleic acid sequencesencoding the antibody of interest, e.g., an antibody described herein,and a signal sequence for secretion. The milk produced by females ofsuch transgenic mammals includes, secreted therein, the antibody ofinterest, e.g., an antibody described herein. The antibody can bepurified from the milk, or for some applications, used directly.

Antibodies can be modified, e.g., with a moiety that improves itsstabilization and/or retention in circulation, e.g., in blood, serum,lymph, bronchoalveolar lavage, or other tissues, e.g., by at least 1.5,2, 5, 10, or 50 fold.

For example, a VLA-4 binding antibody can be associated with a polymer,e.g., a substantially non-antigenic polymer, such as a polyalkyleneoxide or a polyethylene oxide. Suitable polymers will vary substantiallyby weight. Polymers having molecular number average weights ranging fromabout 200 to about 35,000 daltons (or about 1,000 to about 15,000, and2,000 to about 12,500) can be used.

For example, a VLA-4 binding antibody can be conjugated to a watersoluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of suchpolymers include polyalkylene oxide homopolymers such as polyethyleneglycol (PEG) or polypropylene glycols, polyoxyethylenated polyols,copolymers thereof and block copolymers thereof, provided that the watersolubility of the block copolymers is maintained. Additional usefulpolymers include polyoxyalkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethylene andpolyoxypropylene (Pluronics); polymethacrylates; carbomers; branched orunbranched polysaccharides that comprise the saccharide monomersD-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid(e.g. polymannuronic acid, or alginic acid), D-glucosamine,D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; heparin or heparon.

Exemplary Second Agents

In some cases, the formulations described herein, e.g., formulationscontaining an alpha-4 binding antibody, include a second agent, or areadministered in combination with a formulation containing a secondagent.

In one implementation, the α4 binding antibody and second agent isprovided as a co-formulation, and the co-formulation is administered tothe subject. It is further possible, e.g., at least 24 hours before orafter administering the co-formulation, to administer separately onedose of the α4 binding antibody formulation and then one dose of aformulation containing the second agent. In another implementation, theantibody and the second agent are provided as separate formulations, andthe step of administering includes sequentially administering theantibody and the second agent. The sequential administrations can beprovided on the same day (e.g., within one hour of one another or atleast 3, 6, or 12 hours apart) or on different days.

Generally, the antibody and the second agent are each administered as aplurality of doses separated in time. The antibody and the second agentare generally each administered according to a regimen. The regimen forone or both may have a regular periodicity. The regimen for the antibodycan have a different periodicity from the regimen for the second agent,e.g., one can be administered more frequently than the other. In oneimplementation, one of the antibody and the second agent is administeredonce weekly and the other once monthly. In another implementation, oneof the antibody and the second agent is administered continuously, e.g.,over a period of more than 30 minutes but less than 1, 2, 4, or 12hours, and the other is administered as a bolus. The antibody and thesecond agent can be administered by any appropriate method, e.g.,subcutaneously, intramuscularly, or intravenously.

In some embodiments, each of the antibody and the second agent isadministered at the same dose as each is prescribed for monotherapy. Inother embodiments, the antibody is administered at a dosage that isequal to or less than an amount required for efficacy if administeredalone. Likewise, the second agent can be administered at a dosage thatis equal to or less than an amount required for efficacy if administeredalone.

Non-limiting examples of second agents for treating multiple sclerosisin combination with an α4 binding antibody include:

-   -   interferons, e.g., human interferon beta-1a (e.g., AVONEX® or        REBIF®)) and interferon beta-1b (BETASERON™; human interferon        beta substituted at position 17; Berlex/Chiron);    -   glatiramer acetate (also termed Copolymer 1, Cop-1; COPAXONE™;        Teva Pharmaceutical Industries, Inc.);    -   RITUXAN® (rituximab) or another anti-CD20 antibody, e.g., one        that competes with or binds an overlapping epitope with        rituximab;    -   mixtoxantrone (NOVANTRONE®, Lederle);    -   a chemotherapeutic, e.g., clabribine (LEUSTATIN®), azathioprine        (IMURAN®), cyclophosphamide (CYTOXAN®), cyclosporine-A,        methotrexate, 4-aminopyridine, and tizanidine;    -   a corticosteroid, e.g., methylprednisolone (MEDRONE®, Pfizer),        prednisone;    -   an immunoglobulin, e.g., RITUXAN® (rituximab); CTLA4 Ig;        alemtuzumab (MABCAMPATH®) or daclizumab (an antibody that binds        CD25);    -   statins; and    -   TNF antagonists.

Glatiramer acetate is a protein formed from a random chain of aminoacids—glutamic acid, lysine, alanine and tyrosine (hence GLATiramer).Glatiramer acetate can be synthesized in solution from these amino acidsat a ratio of approximately 5 parts alanine to 3 parts lysine, 1.5 partsglutamic acid and 1 part tyrosine using N-carboxyamino acid anhydrides.

Additional second agents include antibodies or antagonists of otherhuman cytokines or growth factors, for example, TNF, LT, IL-1, IL-2,IL-6, IL-7, IL-8, IL-12 IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, andPDGF. Still other exemplary second agents include antibodies to cellsurface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40,CD45, CD69, CD80, CD86, CD90 or their ligands. For example, daclizubmabis an anti-CD25 antibody that may ameliorate multiple sclerosis.

Still other exemplary antibodies include antibodies that provide anactivity of an agent described herein, such as an antibody that engagesan interferon receptor, e.g., an interferon beta receptor. Typically, inimplementations in which the second agent includes an antibody, it bindsto a target protein other than VLA-4 or other than α4 integrin, or atleast an epitope on VLA-4 other than one recognized by the first agent.

Still other additional exemplary second agents include: FK506,rapamycin, mycophenolate mofetil, leflunomide, non-steroidalanti-inflammatory drugs (NSAIDs), for example, phosphodiesteraseinhibitors, adenosine agonists, antithrombotic agents, complementinhibitors, adrenergic agents, agents that interfere with signaling byproinflammatory cytokines as described herein, IL-1β converting enzymeinhibitors (e.g., Vx740), anti-P7s, PSGL, TACE inhibitors, T-cellsignaling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathloprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof, as described herein, anti-inflammatory cytokines (e.g. IL-4,IL-10, IL-13 and TGF).

In some embodiments, a second agent may be used to treat one or moresymptoms or side effects of MS. Such agents include, e.g., amantadine,baclofen, papaverine, meclizine, hydroxyzine, sulfamethoxazole,ciprofloxacin, docusate, pemoline, dantrolene, desmopressin,dexamethasone, tolterodine, phenytoin, oxybutynin, bisacodyl,venlafaxine, amitriptyline, methenamine, clonazepam, isoniazid,vardenafil, nitrofurantoin, psyllium hydrophilic mucilloid, alprostadil,gabapentin, nortriptyline, paroxetine, propantheline bromide, modafinil,fluoxetine, phenazopyridine, methylprednisolone, carbamazepine,imipramine, diazepam, sildenafil, bupropion, and sertraline. Many secondagents that are small molecules have a molecular weight between 150 and5000 Daltons.

Examples of TNF antagonists include chimeric, humanized, human or invitro generated antibodies (or antigen-binding fragments thereof) to TNF(e.g., human TNF α), such as D2E7, (human TNFα antibody, U.S. Pat. No.6,258,562; BASF), CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNFαantibody; Celltech/Pharmacia), cA2 (chimeric anti-TNFα antibody;REMICADE™, Centocor); anti-TNF antibody fragments (e.g., CPD870);soluble fragments of the TNF receptors, e.g., p55 or p75 human TNFreceptors or derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL™; Immunex; see e.g., Arthritis &Rheumatism (1994) Vol. 37, 5295; J. Invest. Med. (1996) Vol. 44, 235A),p55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein (LENERCEPT™));enzyme antagonists, e.g., TNFα converting enzyme (TACE) inhibitors(e.g., an alpha-sulfonyl hydroxamic acid derivative, WO 01/55112, andN-hydroxyformamide TACE inhibitor GW 3333, -005, or -022); andTNF-bp/s-TNFR (soluble TNF binding protein; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), 5284; Amer. J.Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42).

In addition to a second agent, it is also possible to deliver otheragents to the subject. However, in some embodiments, no protein or nobiologic, other than the α4 binding antibody and second agent, areadministered to the subject as a pharmaceutical composition. The α4binding antibody and the second agent may be the only agents that aredelivered by injection. In embodiments in which the second agent is arecombinant protein, the α4 binding antibody and second agent may be theonly recombinant agents administered to the subject, or at least theonly recombinant agents that modulate immune or inflammatory responses.In still other embodiments, the α4 binding antibody alone is the onlyrecombinant agent or the only biologic administered to the subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLE Example 1. Variant Anti-VLA-4 Antibodies are More Potent thanHumanized HP1/2

Anti-VLA-4 antibodies were constructed using the germline frameworkIGKV4-1 (or design L1 and L2) or germline-engineered AAH7033.1 (fordesign L3) for the VL chain and germline framework IGHV1-f for VH. Theseantibodies had fewer back mutations than the humanized HP1/2 antibodydescribed in U.S. Pat. No. 6,602,503.

Heavy Chain Variations

The sequences of three variations of the heavy chain are shown in FIG. 1as Design H0, Design H1 and Design H2. Each design has the CDR's ofmurine HP1/2 grafted into the IGHV1-f framework. Design H0 includes noback mutations of the framework regions, while Designs H1 and H2 havevarious degrees of back mutations in the framework regions sequences tooptimize the affinity of the humanized antibody.

Light Chain Variations

The sequences of four variations of the light chain are shown in FIG. 2as Design L0, Design L1, Design L2 and Design L3 (also called L0, L1,L2, L3). Each design has the CDR's of murine HP1/2 grafted into thegermline framework. The IGKV4-1 germline framework was used for DesignsL0, L1, and L2, and the AAH70335 germline engineered framework was usedfor Design L3. Design L0 includes no back mutations of the frameworkregions, while Designs L1, L2, and L3 have various degrees of backmutations in the framework regions to optimize the affinity of thehumanized antibody.

The results of competition ELISA assays are shown in Table 1 and FIG. 3.In this experiment α4β1 was preincubated with testing mAb and thenmurine HP1/2 was used as competing reagent. The results of thisexperiment indicated that the antibodies having light chains L2 or L3were more potent than the humanized antibody HuHP1/2 described in U.S.Pat. No. 6,602,503. The results are shown in Table 1 below, and in FIG.3. The heavy chain (H1) in the antibodies for this assay had the “DesignH1” sequence shown in FIG. 1, whereas L1 refers to Design L1 in FIG. 2.

TABLE 1 Competition Assay by ELISA mAb IC50 nM Chimeric HP1/2 1.06 H1L01.87 H1L1 1.67 H1L2 0.9 H1L3 0.49 HuHP1/2 1.05

In Table 1, the chimeric mAb is chimerized HP1/2 antibody, where murinevariable heavy and light chains are genetically fused to human IgG1constant regions. This antibody is essentially identical in bindingaffinity to the original murine HP1/2 antibody (Sanchez-Madrid et al.,Eur. J. Immunol. 16:1343-1349, 1996). The results of the experimentindicate that it is possible to improve the affinity of the monoclonalantibody relative to its murine parental sequence through humanizationon germline-engineered acceptor framework.

Another competition assay compares the binding affinity of the newantibodies with the humanized 21.6 anti-α4 antibody (TYSABRI®(natalizumab)) described in U.S. Pat. No. 5,840,299. In this experimentthe binding of mixture of mouse HP1/2 with testing mAb to α4β1 wasassayed. The results of this experiment are shown in FIG. 4 and in Table2 below, and indicate that the newly designed antibodies are about10-fold more potent than natalizumab.

TABLE 2 Competition Assay by ELISA mAb IC50 nM Chimeric HP1/2 1.64 H1L04.46 H1L1 4.55 H1L2 1.34 HuHP1/2 1.41 TYSABRI ® 10.9

Example 2. Humanized HP1/2 (HuHP1/2) Binds VLA-4 on Tumor Cell Lines

Binding of anti-VLA-4 antibody HuHP1/2 to a variety of cell lines wastested by flow cytometry. Binding was tested on CLL (chronic lymphocyticleukemic) cell lines Mec1 and JM1; on MM (multiple myeloma) cell linesU266 and H929; and on AML (acute myelogenous leukemic) cell lines HL60and KG1. HuHP1/2 bound all tumor cell lines tested (FIG. 6). The flowcytometry data was used to calculate the EC50 values for antibodybinding to each of the different cell lines. This information is ahownbelow in Table 3.

HuHP1/2 was also found to block adhesion of AML cell lines tofibronectin (FN) and VCAM1-Ig fusion protein. To test whether theantibody could block adhesion, AML cell lines HL60 or KG1 were allowedto adhere to FN-coated wells (FIG. 7A) or VCAM1-Ig-coated wells (FIG.7B) in the presence of increasing concentrations of HP1/2 or isotypecontrol antibody. HuHP1/2 blocked adhesion of both cell types toFN-coated wells and VCAM1-Ig-coated wells. The maximal inhibition ofHL60 cell binding to both ligands was achieved with 20 μg/ml HuHP1/2(FIG. 7C).

HuHP1/2 was also found to block adhesion of MM cell lines to FN andVCAM1-Ig fusion protein. The MM cell lines U266 and H929 were allowed toadhere to FN-coated wells (FIG. 8A) or VCAM1-Ig-coated wells (FIG. 8B)in the presence of increasing concentrations of HP1/2 or isotype controlantibody. HuHP1/2 blocked adhesion of both types of cell lines to FN-and VCAM1-Ig-coated wells. The maximal inhibition of U266 cell bindingto both ligands was achieved with 20 μg/mL HuHP1/2 (FIG. 8C).

HuHP1/2 was also found to block adhesion of CLL cell lines to FN andVCAM1-Ig fusion protein. The CLL cell lines Mec1 and JM1 were allowed toadhere to FN-coated wells (FIG. 9A) or VCAM1-Ig-coated wells (FIG. 9B)in the presence of increasing concentrations of HP1/2 or isotype controlantibody. HuHP1/2 blocked adhesion of both types of cell lines to FN-and VCAM1-Ig-coated wells. The maximal inhibition of Mec1 cell bindingto both ligands was achieved with 20 μg/ml HuHP1/2 (FIG. 9C).

The IC50 values for HuHP1/2 binding to the tumor cell lines werecalculated from the data shown in FIGS. 7-9. These data are shown inTable 3.

TABLE 3 Quantitation of HuHP1/2 on tumor cell lines IC₅₀ (nM) EC₅₀ (nM)Fibronectin VCAM CLL Mec1 0.11 0.10 0.07 JM1 0.21 — 0.12 MM U266 0.460.14 0.13 H929 0.91 0.21 1.35 AML HL60 0.11 0.16 0.91 KG1 0.19 0.05 0.1

Other embodiments are in the claims

1-29. (canceled)
 30. A recombinant anti-α4 antibody molecule, orα4-binding fragment thereof, comprising: (a) a variable light chaincomprising the amino acid sequence of SEQ ID NO: 11; and (b) a variableheavy chain comprising the amino acid sequence of SEQ ID NO:
 4. 31. Therecombinant anti-alpha4 antibody molecule, or an α4-binding fragmentthereof, of claim 30, comprising a) a light chain constant regionattached to the variable light chain; and b) a heavy chain constantregion attached to the variable heavy chain.
 32. A vector comprising DNAencoding the antibody heavy chain, or α4-binding fragment thereof, andthe antibody light chain, or α4-binding fragment thereof, as defined inclaim
 30. 33. A method of making a recombinant anti-α4 antibodymolecule, or an α4-binding fragment thereof, comprising (a) providing ahost cell comprising a DNA sequence encoding the recombinant antibodymolecule, or an α4-binding fragment thereof, of claim 30 and (b)culturing the cell to produce the recombinant anti-α4 antibody moleculeor α4 binding fragment thereof.
 34. A composition comprising therecombinant anti-α4 antibody molecule, or α4-binding fragment thereof,of claim 30, in combination with a pharmaceutically acceptable carrier.35. A method of treating a patient suffering from a disease or disordermediated by an α4 integrin, comprising administering to said patient thecomposition of claim
 34. 36. The method according to claim 35, whereinsaid patient has cancer.
 37. The method of claim 36, wherein said canceris selected from the group consisting of a solid tumor, a hematologicalmalignancy, a multiple myeloma and acute myelogeneous leukemia (AML).38. The method of claim 35, wherein said disease or disorder mediated byan α4 integrin is selected from the group consisting of an inflammatorydisorder, multiple sclerosis, asthma, rheumatoid arthritis, diabetes,optic neuritis, Crohn's disease, an acute disorder, a spinal cord injuryand traumatic brain injury.
 39. The method of claim 35, wherein thecomposition is administered as a regimen.
 40. The method of claim 35,further comprising administering to the patient a second therapeuticagent.
 41. The method of claim 40, wherein the second therapeutic agentis selected from the group consisting of a thrombolytic agent, achemotherapeutic agent, a neuroprotective agent, an anti-inflammatoryagent, a steroid, a cytokine, and a growth factor.
 42. The methodaccording to claim 36, wherein said cancer is a solid tumor.
 43. Themethod according to claim 35, wherein said disease or disorder mediatedby an α4 integrin is a hematological malignancy.
 44. The methodaccording to claim 35, wherein said disease or disorder mediated by anα4 integrin is multiple myeloma or acute myelogeneous leukemia (AML).45. The method according to claim 35, wherein said disease or disordermediated by an α4 integrin is an inflammatory disorder.
 46. The methodaccording to claim 35, wherein said disease or disorder mediated by anα4 integrin is selected from the group consisting of multiple sclerosis,asthma, rheumatoid arthritis, diabetes, optic neuritis, and Crohn'sdisease.
 47. The method according to claim 35, wherein said disease ordisorder mediated by an α4 integrin is an acute disorder.
 48. The methodaccording to claim 35, wherein said patient is suffering from a spinalcord injury or traumatic brain injury.
 49. The method according to claim35, further comprising administering a second therapeutic agent to thepatient.
 50. The method according to claim 49, wherein the secondtherapeutic agent is selected from the group consisting of athrombolytic agent, a chemotherapeutic agent, a neuroprotective agent,an anti-inflammatory agent, a steroid, a cytokine, and a growth factor.